Despite decades of research, virus infection remains a serious worldwide health threat. Clearly, new ideas and out-of-the-box approaches must be pursued. Many pathogenic viruses, such as influenza, human immunodeficiency, West Nile, and herpes virus, have surface envelopes loaded with carbohydrate-modified proteins encoded by the viral genome. Importantly, these surface glycoproteins are essential for viral infection, so preventing their assembly represents a very promising anti-viral target. The glycoproteins are generally of the N-linked class, which have glycans attached to asparagine residues. Mammalian host cells also synthesize their own N-linked glycoproteins. Thus, to produce their envelope proteins and attach the N-linked glycans, viruses must hijack the host's N-linked glycosylation pathway. This proposal will elucidate a novel anti-viral defense mechanism that prevents this hijacking, and which we hypothesize hinders infection. The antiviral mechanism uses a new signaling pathway elucidated by the P.I. specifically; the P.I. discovered that the common metabolite mannose-6-phosphate (M6P) is generated by glycogen breakdown for double-duty as a 2nd-messenger signaling molecule activated by viral infection. M6P signaling triggers a specific process for destruction of lipid-linked oligosaccharides (LLOs), which in mammals are conjugates of a 14-sugar oligosaccharide (Glc3Man9GlcNAc2) and the carrier lipid dolichol, connected by a pyrophosphate bond. Free Glc3Man9GlcNAc2 and dolichol phosphate result from LLO destruction by M6P signaling. A key point is that since LLOs are the glycan donors for N-linked glycosylation, LLO destruction by M6P signaling is predicted to impair viral N-glycosylation and infectivity. To elucidate this process, four Specific Aims are proposed for years 25-29 of this grant. The experiments will make extensive use of Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), which we adapted for a comprehensive one-stop shopping view of the LLO pool, its substrates, and both its N-glycoprotein and degradation products. Aim I will identify the sensor and effector of M6P, i.e. the proteins that receive M6P's signal, and that carry out M6P's specific instructions for LLO destruction. Aim II will elucidate an unprecedented biosynthetic pathway for M6P 2nd messengers. This pathway is activated by the stress of viral infection, and is distinct from conventional pathways involved in synthesis of M6P as a metabolic precursor. Aim III will identify ER stress-activated components of the pathway. Aim IV will test the effect of M6P signaling on viral infectivity using herpes simplex virus-1 (HSV1). A critical prediction of Aim IV is that M6P signaling will inhibit HSV1 replication. The work will illustrate novel evolutionary aspects of anti-viral defense, and reformat long-standing dogma involving glycogen metabolism. A path for future experiments on the role of M6P signaling in viral pathogenesis and disease will be paved, leading to new anti-viral drugs and treatments.